Project Summary The long-term objective of this project is to discover novel, highly targeted approaches for treating ongoing pain by defining critical mechanisms of ongoing activity (OA) in primary nociceptors that drive this pain. Recent discoveries revealed that the OA generated spontaneously in probable nociceptors and linked to ongoing pain after spinal cord injury (SCI) is associated with all three electrophysiological alterations that, in principle, can promote OA. These are depolarization of resting membrane potential (RMP), reduced voltage threshold for action potentials (APs), and increased frequency of large, transient, depolarizing spontaneous fluctuations (DSFs). Two extrinsic mediators related to inflammation, serotonin (5-HT) and capsaicin (mimicking endogenous TRPV1 activators), also promote OA, in large part by enhancing DSFs. Virtually nothing is known about mechanisms underlying large DSFs. Three specific aims will test hypotheses about DSF generation and potentiation, employing whole cell patch recording, stimulation by Ca2+ uncaging, pharmacological and transgenic approaches, in vivo recording, and behavioral tests. Aim 1 will define ion conductance and cell signaling (Ca2+ and cAMP) contributions to the acute generation of large DSFs, taking advantage of the ability of 5-HT, forskolin, and capsaicin to rapidly stimulate large DSFs, using nave rats and transgenic mice. The focus will include HCN channels, T-type Ca2+ channels, and Nav1.8 channels. Special attention will be paid to TRPC4/5 channels, which are important for OA and have unusual properties that account for unique features of large DSFs. Aim 2 will define ion conductances and cell signals that promote large DSF generation in chronic SCI and in a subacute peripheral inflammation model (hindpaw injection of complete Freund's adjuvant - CFA). The channels found in Aim 1 to be important for large DSFs will be tested for altered contributions and expression in each model. Alterations promoting OA are predicted to be shared in these models (and thus to potentially drive many forms of ongoing pain). Aim 3 will test the prediction that combined interventions selectively blocking large DSFs and elevating AP threshold will reduce ongoing pain. A novel analgesic strategy will be tested, which combines a drug that prevents large DSF generation (a TRPC4/5 blocker) with a drug that selectively elevates AP threshold in nociceptors (a Nav1.8 blocker). The combination should efficiently suppress nociceptor OA and consequent ongoing pain at doses lower than required to observe any effect on ongoing pain from either drug alone. This prediction will be tested in vivo both on C-fiber OA recorded from dorsal roots of anesthetized rats and on ongoing pain in SCI rats and in rat and mouse CFA models. This targeted approach could lay the foundation for new treatments for severe ongoing pain that have relatively few side effects and provide an alternative to opioids, with their attendant risks.